We have hypothesized a critical role for the inflammatory IKKbeta/NF-kappaB pathway in the pathogenesis of obesity- and diet-induced insulin resistance. We now know that the pathway is activated in fat and liver by obesity and high fat diet, that activation of NF-kappaB in fat and/or liver causes insulin resistance, that insulin resistance is transmissible by transplanting affected tissue (fat), and perhaps most importantly, that inhibition of IKKbeta and NF-kappaB, either genetically or pharmacologically, reverses insulin resistance in animals and humans. Elevations in inflammatory markers that are seen in patients are readily reproduced in rodent models of dietary and genetically induced insulin resistance. These are reversed in both rodents and humans in parallel with improvements in insulin resistance, and dramatic reductions in triglyceride, free fatty acid and glucose levels. To continue developing and testing these hypotheses, we now propose a comprehensive plan to identify primary sites of action and tissue-specific effects of genetic up- and down-regulation of NF-kappaB signaling and mechanisms for crosstalk and synergy between tissues. We are activating and inhibiting NF-kappaB selectively in fat, liver, muscle, beta cells and macrophages. Individually, each mouse line will be used to assess the effects on insulin sensitivity and other metabolic parameters. For example, activation of NF-kappaB in fat or liver causes systemic insulin resistance, whereas activation in muscle or beta cells does not. Preliminary results show that selective inhibition of NF-kappaB in fat or liver does the opposite, it prevents insulin resistance from developing even in high fat feeding or ob/ob mice. Additional experiments plan to look at the additive or ameliorative effects of simultaneous activation/inhibition in two tissues. These studies aim to identify a root cause of obesity- and diet-induced insulin resistance and the tissue-specific effects of inhibiting this pathway, and to determine mechanisms of crosstalk and synergy between tissues involved in the development of this very common disorder.